Visualization of single molecules of mRNA in situ.

Publisher Summary The chapter discusses methods and concepts that facilitate the detection and identification of single molecules of mRNA in situ using fluorescence in situ hybridization (FISH). FISH is a very widely used technique in cell biology. The sensitivity of detection is the major concern when implementing the FISH technology. The methodology employs stringent imaging requirements that include a carefully calibrated quantitative epifluorescence digital imaging microscope, three-dimensional (3D) optical sectioning, constrained iterative deconvolution, and 3D interactive analysis software. The analysis of point sources representing one hybridized probe has provided additional information concerning the nature of the imaging process. The pixel does not suffice as the unit for analysis of a digital image involving fluorescence in situ hybridization. The detailed characterization of biological molecules in situ is now possible using FISH. Unprecedented detail can be revealed from a hybridized cell that has been interrogated with carefully engineered, quantitatively accurate probes, followed by three-dimensional imaging and deconvolution.

[1]  Isoform-specific 3'-untranslated sequences sort alpha-cardiac and beta- cytoplasmic actin messenger RNAs to different cytoplasmic compartments [published erratum appears in J Cell Biol 1993 Dec;123(6 Pt 2):following 1907] , 1993, The Journal of cell biology.

[2]  Kiwamu Saito,et al.  Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution , 1995, Nature.

[3]  D. Agard,et al.  The use of a charge-coupled device for quantitative optical microscopy of biological structures. , 1987, Science.

[4]  K. Nasmyth,et al.  Mating type switching in yeast controlled by asymmetric localization of ASH1 mRNA. , 1997, Science.

[5]  W Carrington,et al.  Digital imaging microscopy of living cells. , 1998, Trends in cell biology.

[6]  Robert H. Singer,et al.  Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization , 1989, Cell.

[7]  David M. Shotton,et al.  Electronic light microscopy: present capabilities and future prospects , 1995, Histochemistry and Cell Biology.

[8]  R. Singer,et al.  Movement of nuclear poly(A) RNA throughout the interchromatin space in living cells , 1999, Current Biology.

[9]  S Inoué,et al.  Imaging of unresolved objects, superresolution, and precision of distance measurement with video microscopy. , 1989, Methods in cell biology.

[10]  E. W. Moomaw,et al.  Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes. , 1986, Nucleic acids research.

[11]  J. L. Harris,et al.  Diffraction and Resolving Power , 1964 .

[12]  I T Young,et al.  Image fidelity: characterizing the imaging transfer function. , 1989, Methods in cell biology.

[13]  D. Albertson,et al.  Expression patterns of predicted genes from the C. elegans genome sequence visualized by FISH in whole organisms , 1995, Nature Genetics.

[14]  L. Hood,et al.  The synthesis of oligonucleotides containing an aliphatic amino group at the 5' terminus: synthesis of fluorescent DNA primers for use in DNA sequence analysis. , 1985, Nucleic acids research.

[15]  John J. Lemasters,et al.  Optical microscopy: emerging methods and applications , 1993 .

[16]  David Halliday,et al.  Physics for students of science and engineering , 1960 .

[17]  Lawrence M. Lifshitz,et al.  Coupling of the Na+/Ca2+exchanger, Na+/K+ pump and sarcoplasmic reticulum in smooth muscle , 1993, Nature.

[18]  B. Trask,et al.  Evidence for the organization of chromatin in megabase pair-sized loops arranged along a random walk path in the human G0/G1 interphase nucleus , 1995, The Journal of cell biology.

[19]  R. Singer,et al.  Use of oligodeoxynucleotide probes for quantitative in situ hybridization to actin mRNA. , 1987, Analytical biochemistry.

[20]  D. Agard.  Optical sectioning microscopy: cellular architecture in three dimensions. , 1984, Annual review of biophysics and bioengineering.

[21]  P. Butterworth.  Topics in molecular and structural biology , 1994 .

[22]  B. Trask,et al.  DNA sequence mapping by fluorescence in situ hybridization , 1991, Environmental and molecular mutagenesis.

[23]  E. Betzig,et al.  Near-field spectroscopy of single molecules at room temperature , 1994, Nature.

[24]  D. Albertson,et al.  Fluorescence in situ hybridization for the detection of DNA and RNA. , 1995, Methods in cell biology.

[25]  R. Aikens,et al.  Chapter 16 Solid-State Imagers for Microscopy , 1988 .

[26]  David A. Agard,et al.  Three-dimensional architecture of a polytene nucleus , 1983, Nature.

[27]  F S Fay,et al.  Superresolution three-dimensional images of fluorescence in cells with minimal light exposure. , 1995, Science.

[28]  Robert J. Chichester,et al.  Single Molecules Observed by Near-Field Scanning Optical Microscopy , 1993, Science.

[29]  D. Taylor,et al.  Multiple spectral parameter imaging. , 1989, Methods in cell biology.

[30]  Lawrence M. Lifshitz,et al.  Poly(A) RNA codistribution with microfilaments: evaluation by in situ hybridization and quantitative digital imaging microscopy , 1992, The Journal of cell biology.

[31]  S. Agrawal,et al.  Efficient methods for attaching non-radioactive labels to the 5' ends of synthetic oligodeoxyribonucleotides. , 1986, Nucleic acids research.

[32]  Charles R. Cantor,et al.  Oligonucleotide interactions. III. Circular dichroism studies of the conformation of deoxyoligonucleolides , 1970 .

[33]  R N Zare,et al.  Probing individual molecules with confocal fluorescence microscopy. , 1994, Science.

[34]  K. Kosik,et al.  Sorting of β-Actin mRNA and Protein to Neurites and Growth Cones in Culture , 1998, The Journal of Neuroscience.

[35]  F S Fay,et al.  Visualization of single RNA transcripts in situ. , 1998, Science.

[36]  A. Waggoner,et al.  Cyanine dye labeling reagents containing isothiocyanate groups. , 1989, Cytometry.

[37]  H. Magdelenat,et al.  Principle of digital imaging microscopy. , 1994, Methods in molecular biology.

[38]  F S Fay,et al.  Quantitative digital analysis of diffuse and concentrated nuclear distributions of nascent transcripts, SC35 and poly(A). , 1997, Experimental cell research.

[39]  R. Singer,et al.  Single mRNAs visualized by ultrastructural in situ hybridization are principally localized at actin filament intersections in fibroblasts , 1994, The Journal of cell biology.

[40]  R. Singer,et al.  Neurotrophin Regulation of β-Actin mRNA and Protein Localization within Growth Cones , 1999, The Journal of cell biology.

[41]  J B Lawrence,et al.  Fluorescent detection of nuclear RNA and DNA: implications for genome organization. , 1991, Methods in cell biology.

[42]  F S Fay,et al.  Three‐dimensional molecular distribution in single cells analysed using the digital imaging microscope , 1989, Journal of microscopy.

[43]  R. Singer,et al.  The snoRNA box C/D motif directs nucleolar targeting and also couples snoRNA synthesis and localization , 1998, The EMBO journal.

[44]  Michael R. Green,et al.  Localization of pre-mRNA splicing in mammalian nuclei , 1994, Nature.

[45]  D. Housman,et al.  Foci of trinucleotide repeat transcripts in nuclei of myotonic dystrophy cells and tissues , 1995, The Journal of cell biology.

[46]  R. Singer,et al.  Detection and localization of actin mRNA isoforms in chicken muscle cells by in situ hybridization using biotinated oligonucleotide probes , 1990, Journal of cellular biochemistry.

[47]  B. Trask.  Chapter 1 DNA Sequence Localization in Metaphase and Interphase Cells by Fluorescence in Situ Hybridization , 1991 .